This proposal aims to make lanthanide-based probes available for cellular single molecule microscopy. The objectives are to 1) comprehensively evaluate the feasibility of using time-resolved microscopy to detect single, organic lanthanide complex-labeled biomolecules in mammalian cells;and 2) develop and characterize a general purpose method for the extracellular delivery of lanthanide probes to specific, recombinant fusion proteins. Methodologically, we will adapt epi-fluorescence microscopy for pulsed, near-UV excitation and time-gated, electron-modulated charge coupled device detection. In Specific Aims 1 and 2, we propose to determine the optimal microscopy parameters and the number of LCs per molecule necessary to detect single LC-labeled peptides in vitro and in living mammalian cells, respectively. In Aim 3, we plan to measure the long-term effects of time-resolved microscopy with low-power, pulsed UV excitation radiation on cell motility and DNA structural integrity as well as the immediate effects on sensitive, protein-mediated dynamic processes, including focal adhesion dynamics in adherent fibroblasts, actin dynamics in chemotactic carconoma cells, and GFP-CRAC translocation dynamics in D. discoideium. In parallel, we propose (in Aim 4) to synthetically couple lanthanide probes to a ligand (trimethoprim) that is known to target small molecules to E. coli dihydrofolate reductase (eDHFR) fusion proteins. The targeted probes will be characterized with respect to specificity of labeling, efficacy of transport into and out of cells, and the extent of nonspecific binding or compartmentalization. Successful completion of these aims will determine the extent to which the outstanding photostability and high signal-to-background ratios achievable with time-resolved imaging of lanthanide probes can be applied to time-lapsed imaging of single molecule protein dynamics in living cells. Successful implementation of the trimethoprim-eDHFR targeting strategy will allow for facile specific labeling of selected recombinant proteins. The ability to microscopically detect single molecules and single molecular events inside cells is increasingly important for elucidating the mechanisms of biological function and disease. This proposal seeks to evaluate a promising class of compounds, organic lanthanide complexes, as contrast agents for single molecule microscopy, and to develop a method for selectively labeling proteins in cells with lanthanide probes. In this manner, new tools will be made available to researchers in all areas of biomedical science.